Effector CD8+ T cells are a critical component of the immune system required for the elimination of tumor cells and are the primary target for novel checkpoint inhibitor therapies. Differentiation of naive T cells into effector T cells is characterised by changes in gene expression necessary for T cell function, including expression of cytotoxic molecules such as granzymes and perforin. In addition to regulation by specific transcription factors, effector T cell gene expression is controlled by epigenetic mechanisms such as histone lysine methylation. The histone lysine methyltransferase DOT1L is responsible for methylation of histone 3 at lysine 79 (H3K79). Here, we identify a critical role for DOT1L in the regulation of effector T cell differentiation and function. In vitro, we show that T cell-specific deletion of DOT1L leads to an increase in the number of T cells with an activated phenotype in the early stages of activation, as well as an increase in the number of T cells expressing the effector cytokine IFN-γ and cytotoxic granzymes including granzyme B. Strikingly, we find that DOT1L-deficient CD4+ T cells also express high levels of granzymes and perforin, suggesting that DOT1L has a general role in repression of these pathways in T cells. Furthermore, inhibition of DOT1L in CD8+ T cells using the small molecule inhibitor SGC0946 increases the specific killing of tumor cells in vitro. Collectively, these results demonstrate a pivotal role for DOT1L in regulating effector T cell differentiation and presents DOT1L as a potential target for enhancing T cell responses in cancer immunotherapy.